False-negative CMV PCR results due to viral sequence variation: a diagnostic pitfall with the potential for serious consequences

Background Cytomegalovirus (CMV) continues to be a significant cause of morbidity and mortality in immunocompromised patients. Nucleic acid amplification tests (NAATs) are the preferred method for both diagnosing CMV infection and monitoring response to antiviral therapy in these patients. The use of a sensitive and specific CMV NAAT is essential to ensure early and reliable detection. Case Summary A 4-month-old patient with familial hemophagocytic lymphohistiocytosis received an allogeneic hematopoietic cell transplant (HCT). Weekly CMV monitoring before and after HCT was performed using an in-house quantitative CMV PCR assay that targets the CMV UL54 gene. Review of the amplification curves of PCR runs raised concerns about potential false-negative results. Sequencing of the patient sample identified nucleotide mutations within the probe-binding site, confirming the cause of the assay failure. These false-negative results led to delayed detection of CMV DNAemia before HCT and delayed initiation of CMV preemptive antiviral therapy after transplant. Conclusion This case underscores the critical importance of rigorous and routine evaluation of CMV NAATs by clinical laboratories. Laboratories should recognize the limitations of NAATs and implement strategies to address them. In situations where laboratory findings conflict with clinical data, clinicians should critically assess a negative NAAT result. If CMV infection remains a concern, testing with an alternate PCR assay targeting a different gene should be considered.